Physiologic signaling through the Ras/Raf/MEK/ERK and parallel MEKK/JNKK2/JNK MAP kinase cascades regulates normal epidermal homeostasis, while aberrant activation of these pathways drives epidermal neoplasia. MEK and JNK are activated in a majority of spontaneous human epidermal squamous cell carcinomas, however, two isoforms of each protein are expressed in keratinocytes, and clear functional roles for these pathway elements in human epidermis have not been established. MEK1 null mice are embryonic lethal, precluding establishment of epidermal phenotypes;however, MEK2 null mice are normal. This suggests that MEK1 is either functionally dominant, or functionally redundant with MEK2. The epidermal Ras effects of increased proliferation and decreased differentiation are recapitulated only by MEK1, suggesting a primary role for MEK1. However, MEK1/2 share high homology, and the extent to which MEK2 can compensate for MEK1 loss is unknown. To clarify the relative functional roles of MEK1/2 in human epidermal homeostasis, as well as in epidermal tumorigenesis, we will use complementary RNAi and pharmacologic approaches to inhibit MEK1 and MEK2 in human epidermis. The necessity and sufficiency of MEK1/2 to support Ras driven human epidermal tumorigenesis will then be defined. JNK1 and JNK2 appear to have opposing effects in epidermis. Murine epidermis null for JNK1 is hypoplastic, while JNK2 null epidermis is hyperproliferative. However, the thin hypoplastic JNK1 null tissue is more susceptible than wild type skin to tumor formation, while the hyperproliferative JNK2 null tissue is resistant to tumorigenesis. JNK1/2 roles in normal human epidermis will be defined through RNAi, dominant negative and pharmacologic inhibition. The necessity and sufficiency of JNK1/2 to cooperate with Ras in driving human epidermal tumor formation will then be determined. Aims I and II are based on the hypothesis that MEK1/2 and JNK1/2 are required to support key components of Ras driven epidermal tumorigenesis. At the completion on the proposed funding period, we hope to have defined the importance of MEK1/2 and JNK1/2 signaling in both normal human epidermal homeostasis, and in Ras-driven tumorigenesis as a basis for future therapeutic efforts for human disorders of epidermal growth and differentiation, including cancer.